peptides 27 (2006) 194–210
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
NMR studies for identifying phosphopeptide ligands of the
HIV-1 protein Vpu binding to the F-box protein b-TrCP
Nathalie Evrard-Todeschi a, Josyane Gharbi-Benarous a, Gildas Bertho a,
Gaël Coadou a, Simon Megy a, Richard Benarous b, Jean-Pierre Girault a,*
a
Université René Descartes-Paris V, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques (UMR 8601 CNRS),
45 rue des Saint-Pères, 75270 Paris Cedex 06, France
b
U567-INSERM, UMR 8104 CNRS, Institut Cochin-Département des Maladies Infectieuses, Hôpital Cochin Bat. G. Roussy,
27 rue du Faubourg St-Jacques, 75014 Paris, France
article info
abstract
Article history:
The human immunodeficiency virus type 1 (HIV-1) Vpu enhances viral particle release and,
Received 16 June 2005
its interaction with the ubiquitin ligase SCF-b-TrCP triggers the HIV-1 receptor CD4 degra-
Received in revised form
dation by the proteasome. The interaction between b-TrCP protein and ligands containing
22 July 2005
the phosphorylated DpSGXXpS motif plays a key role for the development of severe disease
Accepted 25 July 2005
states, such as HIV or cancer. This study examines the binding and conformation of
Published on line 13 September 2005
phosphopeptides (P1, LIERAEDpSG and P2, EDpSGNEpSE) from HIV protein Vpu to b-TrCP
with the objective of defining the minimum length of peptide needed for effective binding.
Keywords:
The screening step can be analyzed by NMR spectroscopy, in particular, saturation transfer
Human immunodeficiency virus
NMR methods clearly identify the residues in the peptide that make direct contact with b-
type 1
TrCP protein when bound. An analysis of saturation transfer difference (STD) spectra
Vpu
provided clear evidence that the two peptides efficiently bound b-TrCP receptor protein.
Phosphorylated peptide P-Vpu
To better characterize the ligand–protein interaction, the bound conformation of the
STD-NMR
phosphorylated peptides was determined using transferred NOESY methods, which gave
TRNOESY
rise to a well-defined structure. P1 and P2 can fold in a bend arrangement for the DpSG motif,
Restrained molecular dynamics
showing the protons identified by STD-NMR as exposed in close proximity at the molecule
Bound structure
surface. Ser phosphorylation allows electrostatic interaction and hydrogen bond with the
Binding fragment
amino acids of the b-TrCP binding pocket. The upstream LIER hydrophobic region was also
essential in binding to a hydrophobic pocket of the b-TrCP WD domain. These findings are in
good agreement with a recently published X-ray structure of a shorter b-Catenin fragment
with the b-TrCP complex.
# 2005 Elsevier Inc. All rights reserved.
1.
Introduction
The human immunodeficiency virus type 1 (HIV-1) Vpu
protein, an integral membrane protein of 81 amino acids
(aa) (Fig. 1a), acts as an adaptor for the proteasomal
degradation of CD4. It was shown recently that Vpu exerts a
positive effect on HIV-1 infectivity by down-modulating CD4
receptor molecules at the surface of HIV-1-producing cells [19].
CD4 degradation requires the phosphorylation of the serine
residues at positions 52 and 56 of the Vpu cytoplasmic domain
by casein kinase II [34]. The Vpu secondary structure (Fig. 1b)
consists of one transmembrane helix (h1), connected with a
second helix (h2) residing on the bilayer surface, and a flexible
linker region, including the two phosphorylation sites (Ser52
* Corresponding author. Tel.: +33 1 42 86 21 80; fax: +33 1 42 86 83 87.
E-mail address: [email protected] (J.-P. Girault).
0196-9781/$ – see front matter # 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2005.07.018
peptides 27 (2006) 194–210
195
Fig. 1 – (a) Primary structure sequence of the HIV-1 Vpu protein and (top) sequence of the Vpu fragments (p1 and p2) which
were investigated in this work and (P-Vpu41–62) in a previous work. (b) Domains of the secondary structural regions found in
Vpu. The hydrophobic N-terminal membrane anchor (helix h1, residues 1–28) is followed by two amphipathic a-helices
(helix h2, residues 32–51; helix h3, residues 57–72). Both helices are joined by a flexible non-structured link, which contains
phosphoacceptors Ser52 and Ser56. The C-terminus forms a reverse turn at Ala74 [48]. (c) Part of the backbone (residues 37–
69) of the Vpu cytoplasmic domain (residues 37–81) structure [45].
and Ser56), connects helix h2 with a third helix toward the
C-terminal end [7,10,13,21,37,45,48,49]. Vpu enhances the
release of new virus particles from the plasma membrane
of cells infected with HIV-1 [40] through its N terminal
transmembrane domain (aa 1–27), whereas it induces the
degradation of the CD4 receptor in the endoplasmic reticulum
[39,46] via its cytoplasmic domain (aa 28–81) (Fig. 1c).
Vpu-induced degradation of CD4 requires in fact the
formation of multiprotein complexes [11,35]: Vpu binds to
the F-box b-transducin repeat-containing protein (b-TrCP), the
receptor component of the multisubunit SCFb-TrCP E3 ubiquitin
ligase complex, and connects CD4 to the ubiquitin–proteasome machinery [22]. b-TrCP is a WD40 family F-box protein
(Fig. 2): b-TrCP is linked to the SCF complex by binding to Skp1
through its N-terminal F-box motif and interacts with Vpu
through its C-terminal WD repeat region [22]. b-TrCP is also
involved in the ubiquitination and proteasome targeting of: (i)
b-Catenin, the accumulation of which has been implicated in
various human cancers [12,47], (ii) IkBa, the inhibitor of the
master transcription factor NFkB [16,47,53] and (iii) ATF4, a
member of the family of transcription factors [18]. The signal
for the recognition of all these cellular ligands by b-TrCP is the
phosphorylation of the serine residues present in a conserved
motif, DpSGXXpS for Vpu, IkBa, b-Catenin (Fig. 2c), and
DpSGXXXpS for ATF4. Phosphorylation of Ser52 and Ser56 of
Vpu [31,42] is necessary for the interaction with b-TrCP [34,44].
The SCFb-TrCP complex specifically recognizes a Vpu peptide
fragment of 22 amino acids [22], the P-Vpu41–62 peptide
(Fig. 1a), a 19-amino acid motif in IkBa, and a 22-residue-bCatenin polypeptide in a phosphorylation-dependent manner
[47].
To elucidate the basis of b-TrCP recognition, the bound
structure of the P-Vpu41–62 peptide to the F-box protein b-TrCP
was previously determined by using NMR and MD [8]. This
previous work [8] has allowed us to determine which sequence
requirements are playing a role in interaction with b-TrCP. The
bend region (51–56) corresponding to the DpSGXXpS motif
associated with the hydrophobic cluster (45–46) characterizes
the phosphorylation-dependent recognition by the WD
domain (Fig. 2a). A similar kinked-like DpSGXXpS motif also
plays a central role in the crystal structure of the human bTrCP1-Skp1 complex bound to a fragment b-Catenin substrate
peptide (Fig. 2c) [50].
This study examines the binding and conformation of
phosphopeptides from HIV protein Vpu P1 (P-Vpu45–53) and P2
(P-Vpu50–57) (Fig. 1a) to b-TrCP with the objective of defining the
minimum length of peptide needed for effective binding. The
starting models were chosen with some arbitrary constraints.
The P1 peptide (LIERAEDpS52G), consists of the Vpu residue
pSer52 which was previously found to make the most
196
peptides 27 (2006) 194–210
Fig. 2 – (a) The full-length human protein b-TrCP and below the fusion GST–b-TrCP protein which was investigated in the
present work. This recombinant protein includes the 218 residues of the glutathione S-transferase (GST) protein fused to
the seven WD repeats (residues 251–569) of the full-length protein b-TrCP by an 18 residues linker. (b) The WD repeats were
identified in the primary sequence of b-TrCP from their alignment with the regular expression. The F-box protein b-TrCP
binds the doubly phosphorylated consensus motif DpSGXXpS in IkBa and b-catenin in an analogous manner to Vpu. (c)
Ribbons diagram of the seven b-propeller structure of the WD40 repeat region of the yeast F-box protein b-TrCP. Surface
representation of the top face of the b-TrCP WD40 domain with the bound DpSGIHpS motif from the crystal structure of the
human b-TrCP-SkP1 complex bound to a b-Catenin substrate peptide [50].
extensive b-TrCP contact, and additionally of the amino acids
Leu45 and Ile46 which were found experimentally to be in
close contact with the b-TrCP protein [8]. The P2 peptide
(EDpS52GNEpS56E) contains the majority of the Vpu residues
that were found to be embedded in the b-TrCP WD 40 domain
[50], and located in the bend connecting the helix h2 at the Cterminal. The peptides contain one or two phosphorylated
sites (52 or 52 and 56) for P1 and P2, respectively, and the
residues previously identified for P-Vpu41–62 to be relevant for
binding. The role of these peptides can be investigated by
using STD-NMR epitope mapping and TRNOE-based conformational analysis. These two NMR experiments have taken an
increasing importance as tools in the investigation of
biomolecular recognition phenomena [30]. Saturation transfer
difference (STD) NMR spectroscopy is really well adapted for
analyzing b-TrCP binding processes [14,23,24] by screening
various peptides and mapping of ligand epitopes [25], and the
TRNOESY experiment [28] a fast method to probe the
conformation of a ligand bound to a protein receptor. Finally,
the conformation of the b-TrCP-bound peptides was elucidated by molecular dynamics simulation, an approach
combining dynamical annealing and refinement protocols.
These techniques, described to identify binding events of
ligands to receptors by looking at the resonance signals of the
peptides 27 (2006) 194–210
ligands, allow screening of compounds as well as a detailed
identification of the groups involved in the binding to b-TrCP.
2.
Materials and methods
2.1.
Peptides
HIV-1 encoded virus protein U (Vpu) fragments (residues 45–
53) and (residues 50–57) (numbers refer to Vpu protein), the
peptides P-Vpu45–53 (Ac-Leu-Ile-Glu-Arg-Ala-Glu-Asp-Ser
(PO3H2)-Gly-NH2) named as P1 and P-Vpu50–57 (Ac-Glu-AspSer(PO3H2)-Gly-Asn-Glu-Ser(PO3H2)-Glu-NH2) named as P2,
containing one or two phosphorylated sites (pS52 or pS52
and pS56), with the LIERAEDpS52G and EDpS52GNEpS56E amino
acid sequences, respectively, were purchased from Neosystem
Lab. (Strasbourg, France). The purity of the peptides (95%) was
tested by analytical HPLC and by mass spectrometry.
2.2.
NMR experiments
For the preparation of peptide-b-TrCP samples for NMR
studies, purification of the WD repeat region from human
protein b-TrCP as a fusion protein with the glutathione Stransferase (GST) was described previously [8]. The purified
recombinant protein was concentrated using Amicon Ultra-15
centrifugal filter units from Millipore with a 30-kDa molecular
weight cut-off. The solution was washed in NMR buffer and
concentrated several times, performing a buffer exchange in
order to remove the exceeding reduced glutathione. Protein
concentration was determined using the standard Bradford
and Coomassie Brilliant Blue method, following the OD at
595 nm. The final yield of purified GST–b-TrCP from 1 l of Luria
Bertani medium (LB) was about 1.2 mg. This amount of
purified recombinant protein was used to prepare the NMR
samples. NMR samples contained the P1 or P2 peptides with
the GST–b-TrCP protein. They were prepared in NMR
phosphate-buffered saline (20 mM phosphate, 5% D2O and
0.02% NaN3) at pH 7.2. The protein concentration was
estimated to be 0.05 mM and determined by optical density
measurement at 595 nm on the visible absorption. A 40-fold
ligand excess (2 mM) over binding sites was used throughout
the studies. One control sample was prepared at the same w/v
concentration as was used for the GST–b-TrCP sample, and
containing the binding peptide with the glutathione Stransferase lacking the seven WD domain of b-TrCP protein
(Fig. 2a). The latter corresponds to the 218 residues of the GST
protein and an 18 residues linker.
1
H NMR spectra were recorded at 280 K on a Bruker AMX500 spectrometer using a z-axis gradient. Chemical shifts
assignments (Table 1 and Table S4 in Supplementary data)
referred to internal 3-(trimethylsilyl)propionic acid-2,2,3,3-d4,
sodium salt (TSP-d4). Two-dimensional NMR spectra were
recorded in the phase-sensitive mode using the States–TPPI
method [38]. All experiments were carried out using the
WATERGATE pulse sequence for water suppression [32]. Twodimensional 1H, 1H TOCSY spectra were recorded using a
mlev-17 spin–lock sequence [1] with a mixing time (tm) of 35
and 70 ms, respectively. Typically, spectra were acquired with
256 t1 increments, 2048 data points, and a relaxation delay of
197
1.5 s. Spectra were processed using XWIN-NMR software. All
spectra were zero-filled in F1 spectral dimension to 1024 data
points and a sine bell window function was applied in both
dimensions prior to Fourier transformation.
The one-dimensional (1D) 1H STD-NMR [14,23,24] spectra of
the protein–peptide complexes (Figs. 3 and 4) were recorded at
500 MHz with 2048 scans and selective saturation of protein
resonances at 3 ppm (30 ppm for reference spectra) showing
that by irradiating at d = 3 ppm, the entire protein can be
saturated uniformly and can therefore be efficiently used for
the STD-NMR technique. For the on-resonance irradiation
frequency values around 3 ppm are practical because no
ligand nuclei resonances are found in this spectral region
whereas the significant line width of protein signals still
allows selective saturation. Investigation of the time dependence of the saturation transfer with saturation times from 0.2
to 4.0 s showed that 2 s was needed for efficient transfer of
saturation from the protein to the ligand protons. In order to
achieve the desired selectivity and to avoid side-band
irradiation, shaped pulses are employed for the saturation
of the protein signals. STD-NMR spectra were acquired using a
series of 40 equally spaced 50 ms Gaussian-shaped pulses for
selective saturation (then, a total saturation time was of
approximately 2.0 s) [25], with 1 ms delay between the pulses.
With an attenuation of 50 dB, the radiofrequency field strength
for the selective saturation pulses in all STD-NMR experiments
was 190 Hz. The irradiation yields full saturation of the protein
by efficient spin diffusion. Subtraction of FID values with onand off-resonance protein saturation was achieved by phase
cycling. For STD experiments (Figs. 3 and 4) the ligand to
protein ratio was raised to 40:1 (0.2 mM peptide, 50 mM GST–bTrCP protein).
Transferred nuclear Overhauser effect (TRNOESY) [6]
spectra of the b-TrCP–peptide complex were recorded with
4 K points and 512 t1 increments, and a relaxation delay of
1.5 s. Data processing was performed by zero filling to 1 K
points in F1 to give a final 4 K 1 K matrix followed by
multiplication with a squared cosine function and Fourier
transformation. The bound and free states exist in the same
regime, namely vtc > 1; thus the cross-relaxation rates of the
P1 and P2 peptides (MW = 1110 and 1066 g/mol) in the free and
bound state are negative in sign. The free molecules exhibit
very small negative NOEs. The optimal conditions for the
TRNOESY measurements were determined by considering a
peptide:b-TrCP molar ratio ranging from 10 to 100 with mixing
times (tm) of 100, 200, 400 and 500 ms. The buildup curve [17]
for different NOE correlations showed that spin diffusion was
negligible for a tm of 200 ms. After optimization of the peptide:
protein ratio, the final NMR sample was prepared with 3 mg of
GST–b-TrCP protein (0.05 mM) and 1.1 mg of peptide (2 mM),
which corresponds to a peptide:binding site ratio of 40:1, in
500 ml of buffer solution at pH 7.2. The observed TRNOE
correlations with mixing times of 100 and 200 ms were large
and negative. They were assigned to the b-TrCP-bound ligand
[9] as a sample of the peptide without the presence of b-TrCP
protein exhibited only intra-residue and sequential NOE
intensities with a mixing time of 200 ms. Integration of
cross-peak volumes was performed using FELIX software
(Accelrys). Cross-peak intensities were converted to interproton distances using the distance between the Ile CbH2
198
peptides 27 (2006) 194–210
Table 1 – 1H and
13
C NMR chemical shifts of the P1 and P2 peptides bound to the protein b-TrCP in ppm from TSP-d4a
Residue
dNH
dHa
(CH3CO)
Leu 45
Ile 46
Glu 47
Arg 48
Ala 49
Glu 50
Asp 51
pSer 52
Gly 53
(CONH2)
8.36
8.32
8.67
8.54
8.69
8.61
8.44
9.21
8.81
4.30
4.19
4.29
4.35
4.33
4.27
4.65
4.42
3.92
dHb
dHg
dHd
dOthers
1.61
1.50; 1.21
2.25
1.64
0.95; 0.88
0.91; 0.89
dCa
dCb
dCg
dCd
55.0
60.4
55.3
55.4
52.1
56.2
53.7
58.2
44.9
42.2
38.6
30.2
30.9
19.1
30.0
41.2
65.4
26.8
26.8
36.0
26.5
24.5; 23.3
17.1; 12.2
2.03
1.56
1.85
1.93;
1.85;
1.42
1.93;
2.76;
4.10;
2.01
1.77
2.05
2.72
4.15
3.22
6.61; 6.99; 7.37
2.29
43.0; 42.1
35.8
7.20; 7.45
Residue
dNH
dHa
dHb
(CH3CO)
Glu 50
Asp 51
pSer 52
Gly 53
Asn 54
Glu 55
pSer 56
Glu 57
(CONH2)
8.51
8.57
9.03
8.72
8.35
8.70
8.86
8.82
4.31
4.69
4.51
4.00
4.80
4.34
4.53
4.24
2.10; 1.91
2.77; 2.69
4.12
dHg
dOthers
dCa
dCb
dCg
56.6
54.1
58.2
45.6
–
56.5
57.6
56.7
30.3
41.5
65.7
36.1
2.06
2.86;
2.09;
4.11;
2.12;
2.76
1.98
4.02
2.00
2.30
7.88; 7.06
2.32
2.35; 2.32
39.4
30.3
65.8
30.3
36.1
36.1
7.17; 7.68
a
Spectra were recorded at 280 K, pH 7.2, P-Vpu/b-TrCP = 10 and 20 mM sodium phosphate buffer, H2O:2H2O 9:1 (v/v). Resonances marked by a
dash were not visible.
protons (1.76 Å) and between the Asn NH2 protons (1.7 Å) as a
reference for P1 and P2, respectively (Table S5 in Supplementary data). TRNOE cross-peaks classified as strong, medium,
and weak were converted into distance restraints of 1.8–2.7,
1.8–3.6 and 1.8–6.0 Å, respectively.
2.3.
Structure calculations
Distance restraints used in the structure calculations were
derived from TRNOESY experiments performed with mixing
times of 100 and 200 ms, and as for the free ligand, we obtained
a blank TRNOESY spectrum at 100 ms and only intra-residue
and sequential NH-Ha correlations at 200 ms. The final list of
distant restraints (Table 2) containing 75 unambiguous and 12
ambiguous (42 intra-residue, 26 sequential, 10 medium-range
and 9 long-range) restraints for P1 and 86 unambiguous and 6
ambiguous (36 intra-residue, 13 sequential, 20 medium-range
and 17 long-range) restraints for P2 was incorporated for
structure calculation with the standard protocol of ARIA 1.2
[4,20]. Modifications of the phosphorylated residues (pSer)
were introduced with CHARMM [3] for molecular dynamics
calculations using the PARALLHDG 5.3 force field. During the
calculations, non-glycine residues were restrained to negative
f values (usually the only range considered in NMR-derived
structures) [33]. The simulated annealing protocol consisted of
four stages: a high-temperature torsion angle simulated
annealing phase at 10,000 K (30 ps), a first torsion angle
dynamics cooling phase from 10,000 to 50 K (15 ps), a second
Cartesian dynamics cooling phase from 2000 to 50 K (27 ps),
and a final minimization phase at 50 K. ARIA enabled the
incorporation of ambiguous distance restraints and calibra-
tion of the NOE restraints using automated matrix analysis as
implemented by the program. To differentiate between the
probable contributions for each of the ambiguous NOEs, the
automated assignment program ARIA was used. Various runs
were performed to utilize as many unambiguous and
ambiguous restraints as possible from the 500 MHz H2O
TRNOESY spectra. ARIA runs were performed using the
default parameters with eight iterations. Twenty structures
were generated each round, and the 10 lowest-energy
structures were carried on to the next iteration. In the final
iteration, the 20 lowest-energy structures were retained as the
final structures. For the free peptides only, a procedure with
explicit solvent molecules incorporated during the final run
was selected using a box of water molecules with ARIA
software (OPLS force field). It was decided that due to the
presence of the bound peptide in the hydrophobic recognition
sites this step was not applicable in the bound state. The set of
P1 and P2 structures was selected for correct geometry and no
distances restraint violations of >0.5 Å. Analysis of the
structures was performed within Aqua, procheck-NMR (30)
programs (Table 2). MOLMOL [15] was used for the analysis and
presentation of the results of the structure determination
(Table 3).
3.
Results
The recombinant protein includes the 218 residues of the GST
protein fused to the seven WD repeats (residues 251–569) of
full-length protein b-TrCP by an 18 residues linker. The GST
fusion partner, indeed, enhances the solubility and stability of
peptides 27 (2006) 194–210
199
Fig. 3 – (a) 1D 1H spectrum (bottom) and 1D 1H STD-NMR spectrum (top) of the P1 peptide in association with the GST–b-TrCP
protein, showing enhancements of resonances of protons making close contacts with the protein combining site; the
GST–b-TrCP protein displayed few broad signals (asterisk) at 1.0–3.0 ppm. (b) Expansion of the region containing
resonances of the amide protons.
the WD domain and provides a method of immobilizing the
protein on a glutathione-derived matrix. The obtained protein,
which was used to perform NMR interaction experiments, is
hereafter referenced as GST–b-TrCP, and its molecular weight
is approximately 60 kDa.
3.1.
1
NMR resonance assignments
H chemical shifts and resonance assignments were established using two-dimensional 1H,1H TOCSY and NOESY
experiments [51] and are reported in Table 1 and Table S4
in Supplementary data. Sequential assignments of 1H resonances were based on characteristic sequential NOE connectivities observed between the a-proton of residue i and the
amide proton of residue i + 1, i.e. daN (i, i + 1) in NOESY data set.
Upon binding of a ligand to a receptor protein, the chemical
shifts of both the ligand and protein proton resonance signals
are affected. Addition of GST–b-TrCP caused a line broadening
of the P1 and P2 signals, and a chemical shift difference
relative to the free peptides (Fig. S11 in Supplementary data),
thus providing a clear indication of the existence of binding.
Interestingly, in the presence of b-TrCP protein, a slight lowfrequency (shielded) shift of the Ha protons was observed in
the 47–52 N-terminal region of P1 and in the 52–57 region of the
P2 peptide. An opposite shift was observed with a large high-
frequency (unshielded) shifted HN resonance, particularly for
pSer52 in both peptides, and a slight high-frequency shift for
Glu47, Arg48, Asp51 in P1 peptide and for pSer56, Glu57 in the
P2 peptide. These shifts may be an indicator of intermolecular
contact of the peptides with the binding site. The interaction
caused environmental changes on the peptide protein interfaces and hence, affected the chemical shifts of the nuclei in
this area. Fast exchange was measured for the interaction of
the phosphorylated peptides to the GST–b-TrCP protein with a
dissociation constant estimated between 500 mM and 1 mM
[28,43]. This range of binding affinity made the peptides likely
to be suitable for TRNOESY NMR experiments, which require
fast exchange between the free and bound states.
3.2.
b-TrCP binding site for P1 and P2
To provide additional information regarding the peptide mode
of binding, STD-NMR experiments were performed [14,23,24].
The STD-NMR experiment was initially applied to screen small
peptides of Vpu protein for binding activity towards the b-TrCP
binding protein. Resonances of the protein are selectively
saturated, and in a binding ligand, enhancements are
observed in the difference (STD-NMR) spectrum resulting
from subtraction of this spectrum from a reference spectrum
in which the protein is not saturated [14,23,24]. Protons of the
200
peptides 27 (2006) 194–210
Fig. 4 – (a) 1D 1H spectrum (bottom) and 1D 1H STD-NMR spectrum (top) of the P2 peptide in association with the GST–b-TrCP
protein, showing enhancements of resonances of protons making close contacts with the protein combining site; the
GST–b-TrCP protein displayed few broad signals (asterisk) at 1.0–3.0 ppm. (b) Expansion of the region containing
resonances of the amide protons.
ligand, which are in close contact with the protein, can easily
be identified from the STD-NMR spectrum, because they are
saturated to the highest degree. They should have stronger
STD, and this allows direct observation of areas of the ligand
that comprises the epitope.
With regards to the spin diffusion, in the range of the
saturation times that we used (from 0.2 to 4.0 s), we observed
similar relative results for all the protons. The global intensity
of all the signals was modified consistently, indicating that
there was no visible spin diffusion under these conditions. It
has also been shown that epitope mapping is possible if the
ligand can leave the binding site before all magnetization has
been equally distributed among all spins in the ligand [24]. In
this case, we can say that there is no visible spin diffusion
within the peptide. This condition is fulfilled for ligands with a
high turnover number. Since (i) we work with a large excess of
peptide, and (ii) our peptide is a weakly binding ligand with
dissociation constant in the range of the mM, the turnover
number, or dissociation rate constant, is high enough to map
the interaction. For this reason, we do not observe spin
diffusion within the peptide.
We investigated the interaction of the P1 and P2 peptides
with b-TrCP protein. The resulting STD-NMR spectra in the
presence of the protein clearly indicated the binding of the two
peptides. Figs. 3 and 4 shows 1D STD spectra and normal 1H
spectra of the complexes of P1 and P2 with the b-TrCP protein.
1D 1H NMR spectrum of P1 and P2 in the presence of the
protein displayed few broad signals at 3.0 and 1.0–1.5 ppm,
normal for a protein.
The different signal intensities of the individual protons
are best analyzed from the integral values in the reference (I0)
and STD spectra (I0 Isat), respectively. The integral value of
the largest signal of P1 and P2 peptides, the Asp51 H–N
proton, was set to 100% (Fig. 5). The relative degree of
saturation for the individual protons normalized to that of
the Asp51, can be used to compare the STD effect [24]. The 1D
spectra of the P2 peptide in a 40-fold excess over the GST–bTrCP protein show clearly that the pSer52 (41%) and pSer56
(77%) H–N resonances, and other DpSGNEpS motif resonances belonging to Glu50 (52%), Asp51 (100%), Gly53 (60%),
Asn54 (87%), Glu55 (60%), or Glu57 (51%) have STD intensities
between 50 and 100% (Fig. 5). Thus, they are distinctly
involved in binding.
On the other hand, the functional groups involved in the
binding of the ligand P1 to the GST–b-TrCP protein, the H–N
resonances of Leu45 (62%), Ile46 (56%), Arg48 (58%) and Ala49
(74%) have similar larger STD intensities, ranging from 50 to
75%, indicating that these residues are involved in direct
binding to the protein, in addition to Glu50 (84%), Asp51 (100%),
pSer52 (78%) and Gly53 (68%).
Interestingly, for P1 and P2 peptides, the amino acids in
common, Asp51, pSer52 and Gly53 received a similar fraction
201
peptides 27 (2006) 194–210
Table 2 – Structural statistics of the final 10 NMR structures of P-Vpu peptides, P1 and P2 bound to the b-TrCP protein
ARIAoutput
P1
No. of experimental distance restraints
Unambiguous NOEs
Ambiguous NOEs
Total NOEs
Intra
Sequential
Medium-range
Long-range
P2
75
12
87
42
26
10
9
86
6
92
36
13
20
17
8
6
0.04 0.01
0.04 0.01
0.04 0.01
0.03 0.001
0.01 0.003
0.03 0.002
NOE violations >0.5 Å
NOE violations >0.3 Å
0
0
0
0
RMS differences from mean structureb (Å)
Backbone
Heavy
0.55 0.16
1.73 0.61
0.55 0.20
1.48 0.37
Ramachandran plot of residuesc
In most favored regions
In additional allowed regions
In generously allowed regions
In disallowed regions
71
29
0
0
60
40
0
0
No. of experimental broad dihedral restraints
RMS differences from distance restraints
Unambiguous NOE (Å)
Ambiguous NOE (Å)
All NOEs (Å)
a
b
c
a
Calculated by ARIA.
Calculated by MOLMOL.
Calculated by PROCHECK.
of saturation. The saturation transfer also presents maximum
intensity for the acetyl protons of N-terminal and the NH
amide protons of C-terminal (Figs. 3 and 4). This indicated the
proximity of these protons to the protein surface. For the side
chains of Arg48 (Fig. 3), and Asn54 (Fig. 4), the signals of the NH
protons have similar large STD intensities. The signals
observed in the 1D STD-NMR spectra revealed that the whole
P1 and P2 peptides could be in intimate contact with the
protein.
Optimization of the experimental set-up for STD-NMR
spectroscopy was achieved using samples without GST–bTrCP protein. In that case, STD spectra did not contain ligand
Table 3 – Secondary structure analysis and dihedral angles for the bound structures with lowest energy calculated by MD
calculations with ARIA, for the P1 and P2 peptides in complex with the protein b-TrCPa
Residue
Structure
f
c
x1
Leu45
Ile46
Glu47
Arg48
Ala49
Glu50
Asp51
pSer52
Gly53
Coil
Turn
Turn (helix_3_10, helix1)
Turn (helix_3_10, helix1)
Turn (helix_3_10, helix1)
Bend
Bend
Coil
Coil
91.0
156.4
67.7
75.3
140.8
62.2
87.6
54.6
83.4
164.1
162.5
8.4
1.3
49.4
44.9
46.2
63.6
–
163.5
37.9
57.9
67.4
176.5
55.9
170.1
66.6
–
Glu50
Asp51
pSer52
Gly53
Asn54
Glu55
pSer56
Glu57
Coil
Bend
Bend
Bend
Bend
Bend
Coil
Coil
61.9
151.1
60.2
169.8
66.9
146.6
65.3
164.5
55.8
163.0
14.9
3.6
36.7
65.3
89.0
–
50.0
177.0
35.9
–
177.7
77.8
11.6
176.5
a
The program MOLMOL was used for the analysis and presentation of the results of the structure determination.
202
peptides 27 (2006) 194–210
Fig. 5 – Mean STD values (in percent) of the amide protons
of the individual amino acids calculated for each amino
acid of P1 and P2 from the 1D spectrum.
signals, because saturation transfer does not occur without
the protein. Another experimental way to distinguish here
between specific effects of binding peptide to its target and
non-specific interactions between ligand and macromolecular
complex, was to use in control experiments, the binding
peptides, P1 and P2 with the glutathione S-transferase lacking
the seven WD domain of b-TrCP protein. However, the Cterminal fragment of b-TrCP with the seven WD repeats was
required for binding to Vpu. Thus, the negative control
provided by recording STD-NMR experiments in the presence
of GST protein show clearly the specific binding of the peptide
ligands, P1 and P2 to the GST–b-TrCP protein.
3.3.
Fig. 6 – (a) Sequential dNN(i, i + 1), daN(i, i + 1), dbN(i, i + 1),
dab(i, i + 1) and medium-range daN(i, i + 2), daN(i, i + 3),
dab(i, i + 3) and daN(i, i + 4) 1H–1H TRNOE connectivities in
the peptides P1 and (b) P2 (sequence at the top) in the
presence of the GST–b-TrCP protein at 280 K and pH 7.2.
The thickness of the lines reflects the relative intensities
of the NOEs within the individual plots.
Bound conformation of P1 and P2
TRNOESY experiments [5,6] were used to investigate the
bound conformation of the peptides. The free peptides
exhibited only small negative intra-residue and sequential
NH–Ha correlations, when the mixing times was 200 ms and
negative NOE connectivities only for long mixing times
(tm 500 ms) in aqueous solution and in the absence of bTrCP protein. This fact is in agreement with their conformational studies showing that these peptides do not adopt any
favored structures at all, free in solution.
The molar ratio of P1 and P2 ligand molecules to b-TrCP
receptor protein was investigated from 10 to 100, and the
optimal conditions for the TRNOESY measurements were
observed for a ratio near 40:1. Multiple connectivities are
negative (TRNOEs) and can be assigned to the bound
conformations of low-molecular weight peptides. Since the
peptides are in fast exchange on the cross-relaxation time scale
of the bound peptides, the observed TRNOESY intensities are
sums of the bound and free peptide NOEs. Fast exchange was
measured for the interaction of the phosphorylated peptides to
the GST–b-TrCP protein with a dissociation constant estimated
between 500 mM and 1 mM. In this study, analysis of TRNOEs
was simplified since NOEs, which were present for the free
peptides, were smaller than those for the bound state. With
short mixing times (100–200 ms), free peptide NOEs represent
little uncorrected NOEs because of the longer mixing times
(500–600 ms) used in the free state analysis.
A negative control with only sequential NH–Ha correlations
provided by recording NOESY spectra of the peptides in the
presence of GST protein showed that the large number of
negative NOEs observed in the presence of the fusion GST–bTrCP protein was due to transfer from the bound peptides
(transferred NOE).
A summary of sequential d(i, i + 1) and medium range d(i,
i + 2) and d(i, i + 3) 1H–1H TRNOE connectivities is presented in
Fig. 6 and Table S5 in Supplementary data. In the P1 TRNOESY
spectra, it is interesting to show the propensity for a turn I46–
A49 region. The pattern of TRNOE connectivities in the bound
P1 TRNOESY spectra suggests that a turn is present in the Nterminal portion of the peptide. Turn structure in this segment
is implicated by the density of daN, I46–E47, E47–R48, R48–A49
and dNN, E47–R48, sequential TRNOEs, and the strong longrange TRNOE such as aN(i, i + 3), aN(I46, A49). Side chain-side
chain TRNOEs between the bCH2 protons of Glu47 and gCH2
protons of Leu45 and Ile46 and between the bCH2 protons of
Arg48 and bCH2 protons of Glu47 and Ala49 have been
observed. On the other hand, the presence of medium
aN(i, i + 1), aN(R48, A49), aN(A49, E50), aN(E50, D51), aN(D51,
pS52), aN(pS52, G53) cross-peaks, intense bN(i, i + 1), bN(A49,
E50) connectivities and medium long-range aN(i, i + 4), aN(A49,
G53), cross-peak indicated that the 49–53 region presents
predominantly a bend structure.
The P2 TRNOE spectrum exhibits a great number of NOEs
including intense NN(E55, pS56), medium NN(pS52, G53),
peptides 27 (2006) 194–210
NN(G53, N54) NN(N54, E55) and weak NN(D51, pS52), NN(i, i + 1)
connectivities (Fig. 6) suggesting the presence of secondary
structures (a- or b-turns) for the DpSGNEpS motif. The
presence of intense aN(i, i + 1), aN(i, i + 3) and intense ab(i,
i + 3), TRNOE connectivities, in addition to medium range bN(i,
203
i + 1), aN(i, i + 2) and aN(i, i + 4), TRNOE connectivities also
denote the presence of secondary structures. These peaks
argue in favor of a folded structure for the DpSGNEpS
sequence, which includes the pSer-phosphorylated site. The
structure was well defined in the region from Glu50 to Glu57,
Fig. 7 – NMR TRNOE-derived structures of the bound peptides in the presence of GST–b-TrCP protein. (a) Superimposition of
10 structures generated after eight iterations with ARIA software for P1 and (b) for P2. (c) Energy minimized conformer with
the best fit of proton distance constraints for P1 and (d) for P2.
204
peptides 27 (2006) 194–210
reasonable turn motif (Fig. 7a and c). The motif L45IERA49 has
a stabilizing effect on the helical conformation within
distances of less than 0.2 nm between each of the residues,
and with two hydrophobic residues, Leu45, Ile46 preceding
the charged Glu47Arg48 motif. Arg48 is further able to support
the loop via the formation of salt bridge with Glu50 and
Asp51.
On the other hand, TRNOE NMR studies of P2 peptide in the
presence of GST–b-TrCP protein showed that the bound
conformation of the peptide is a bend conformation from
residues Asp51 to Glu57 (Fig. 7b and d). This bend would
expose the side chains of residues 51–57 for specific interactions with the b-TrCP protein combining site that is consistent
with the STD-NMR data, showing enhancements of the
residues in the same region, between 50 and 100% (Fig. 5),
and with the chemical shift variation involving residues of this
loop region (Fig. S11 in Supplementary data).
where a bend was apparent. Strong aN contact between E50D51, D51-pS52, G53-N54, N54-E55, pS56-E57, G53-pS56, medium aN contact G53-E55, D51-E55, pS52-pS56 and other
interresidue contacts such as ab(G53, pS56), and bN(N54,E55),
bN(E55,S56), helped to define this bend.
To study the conformation of the bound state of P1 or P2 in
the presence of the GST–b-TrCP protein, the distance
restraints were incorporated into a simulated annealing
protocol using ARIA. The structures that were generated
resulted in NOE restraint files consisting of 87 and 92
restraints, for P1 or P2, respectively (Table 2). A set of 20
structures produced by simulated annealing was subjected to
energy minimization, followed by checks for correct geometry
and agreement with the distance restraints (Fig. 7). The
structural models fit the NMR data well, with no violations of
experimental distance restraints greater than 0.3–0.5 Å. The
positions of the backbone (Table 3) and most side chain atoms
were well defined by the NMR restraints. Structural statistics
are presented in Table 2. Structure calculations by simulated
annealing using NOE constraints followed with refinement
and energy minimization led to the family of 10 structures
shown in Fig. 7a and b for P1 and P2, respectively. TRNOE NMR
studies of P1 peptide in the presence of GST–b-TrCP protein
showed, for the bound conformation of the peptide, a
propensity for turn formation in N-terminal residues 46–49,
and a short bend including the EDpS52 part of the phosphorylation motif with the phosphate group pointing away (Fig. 7c;
Table 3). The P2 calculated structures (Fig. 7d; Table 3)
comprise a large bend from Asp51 to Glu55. These structures
would expose the pSer side chains for interaction with the
GST–b-TrCP protein, a hypothesis consistent with the STDNMR data. The average root mean square difference for
superimposition of the 10 structures with the lowest NOE
restraint energy was 0.4 Å for the backbone atoms (N, Ca, C, O)
of residues 51–55. The 10 P2 structures superimposed with
RMSD = 0. 55 Å for backbone atoms considering the entire
peptide. The 10 structures of the P1 peptide (Table 2) superimposed with the same RMSD.
The SCFb-TrCP complex specifically recognizes a Vpu peptide
fragment of 22 amino acids [22], the P-Vpu41–62 peptide
(Fig. 8a), of which the bound structure to the b-TrCP protein
was previously determined by NMR and MD [8]. After superimposition of the two bound P1 and P2 peptides and the
corresponding fragments in the b-TrCP-bound P-Vpu41–62
peptide (Fig. 8b and c), the RMSD values of their backbone
coordinates are 0.35 and 0.31 Å, respectively. The bend
(DpSGNEpS) is similarly found in the b-TrCP-bound P2 and
P-Vpu41–62 peptides [8], as shown in Fig. 8c. The phosphorylated pSer52 residues would then be able to dock to b-TrCP
(Fig. 8b and c) while the pSer56 phosphate groups are pointing
away involving probably large dynamical motion (Fig. 8c).
In the same way, the turn (LIERAEDpSG) was observed in
the P1 and P-Vpu41–62 peptides bound to b-TrCP (Fig. 8b). The
bound conformation of the P1 peptide falls partly into the
same regular a-helix h2 of the bound phosphorylated peptide,
P-Vpu41–62. Then, their pSer52 residues are fitted and are free to
point into protein combining site.
4.
4.1.2. Comparison with the crystal structure of the complex
b-TrCP-b-Catenin peptide
Discussion
By interfering with cellular proteins such as b-TrCP, Vpu
probably has a major effect on various functions and signaling
pathways in HIV-1-infected cells. As b-TrCP also controls
essential cellular signaling pathways by degrading b-Catenin
and IkBa substrates via the ubiquitin–proteasome system, it
was recently shown that Vpu is a competitive inhibitor of bTrCP that impairs the degradation of SCFb-TrCP substrates as
long as Vpu has an intact DpSGNEpS phosphorylation motif
and can bind to b-TrCP protein [2]. The two ligands with
moderate affinity, P2 peptide, centered on the doubly
phosphorylated motif and P1 peptide, are essential tools in
defining the interactions of Vpu.
4.1.
Bound conformation of the P1 and P2 peptides to the
protein b-TrCP
From TRNOE NMR studies and the P1 simulations it can be
concluded that the residues involved in the peptide retain a
4.1.1.
Comparison with the longer P-Vpu41–62 peptide
We highlighted that interaction of the P2 small peptide relies
on the DpSGXXpS motif, similar to that found in the other
substrates of b-TrCP (entire P-Vpu, IkBa and b-Catenin).
Indeed, the b-turn motif plays a central role in the crystal
structure (Fig. 9a) of the human b-TrCP1-Skp1 complex
bound to a fragment b-Catenin substrate peptide (Fig. 2c)
[50]; of the b-Catenin motif in the crystals, only an 11 residue
segment (residues 30–40), centered on the doubly phosphorylated motif (DpS33GIHpS37), makes the largest number
of b-TrCP contacts (Fig. 9a). As the hydrophobic region
upstream of this motif is not present in the crystal structure,
its possible participation in the binding was not observed.
Therefore, it was interesting to highlight the similarity
between the two bound P1 and P2 peptides and the
corresponding fragment in the b-TrCP-bound b-Catenin
peptide (Fig. 9b and c); after superimposition of the DpSG
motif of the two P1 and P2 peptides and the corresponding
X-ray fragment, the RMSD values of their backbone coordinates are 0.42 and 0.40 Å, respectively. In the P1 and P2
peptides 27 (2006) 194–210
205
Fig. 8 – (a) NMR TRNOE-derived structure of the P-Vpu41–62 bound peptide in the presence of GST–b-TrCP protein. (b)
Superimposition of the NMR bound peptides P-Vpu41–62 and P1. The structures are fitted from residue 46 to 52. (c)
Superimposition of the NMR bound peptides P-Vpu41–62 and P2. The structures are fitted from residue 51 to 55.
peptides, the phosphoserine, aspartic acid, and hydrophobic
residues of the DpSG motif are recognized directly by
contacts from b-TrCP. Again, it appears that the DpSG
segment forms a bend very preserved with a reduced
mobility (Fig. 9b and c) whereas the final part of the
phosphorylation motif (XXpS) seems more mobile.
4.2.
b-TrCP binding site for P-Vpu
4.2.1.
P1 and P2 binding region
The STD-NMR studies of the P2 peptide in the presence of bTrCP showed the involvement of the NH groups of the sixresidue sequence DpSGXXpS in binding. The NH and aliphatic
groups interact strongly with the corresponding amino acids
inside the paratope (STD intensities between 50 and 100%). In
addition, for P1 peptide, the NH and aliphatic groups of Leu45,
Ile46 residues are recognized (STD intensities, ranging from 55
to 65%). This result shows clearly that the P1 also carries a
small portion of binding specificity of the entire Vpu ligand.
The signal intensity of the P1 protons is compared to the
increase of the signals for P2. The relatively similarity in STD
effect (STD intensities between 50 and 100%) shows clearly
that P1 and P2 ligands bind strongly to the receptor protein
while making a distinction in the binding pocket between the
proton involved from the different residues (Fig. 10). The
known factor in binding is the phosphate group of the pSer
residue but the Asp, Glu, Arg and Asn residues also have a
strong contact with the protein. In the case of Asp and Glu, the
contact is through the carboxyl groups whereas in the case of
the Leu or Ile, it is a hydrophobic contact. The Gly also
participates in binding for the two peptides with similar larger
STD intensities, ranging from 60 to 68% (Fig. 5).
206
peptides 27 (2006) 194–210
Fig. 9 – (a) Close-up view of the doubly phosphorylated DpSGIHpS motif bound, from the crystal structure of the human
b-TrCP-SkP1 complex bound to a b-Catenin substrate peptide and recognized by the F-box protein b-TrCP [50].
Superimposition of the DpSG motif of the P-b-catenin30–40 X-ray crystal structure and the corresponding residues (b) of
the P1 bound structure and (c) of the P2 bound structure.
The NMR data described above show that the epitope
comprises a surface extending over residues of the bend
DpS52GNEpS56 motif of the bound P2 peptide associated with
the hydrophobic cluster (Leu45–Ile46). Leu45 and Ile46, whose
hydrophobic nature is conserved in the P1 peptide fragment,
are able to make van der Waals contacts with a hydrophobic
pocket that would be composed of the aliphatic portion of the
Val516 and Phe523 side chains in b-TrCP (Fig. 10a). This turn
hydrophobic region is able to project Asp51 and pSer52 into a
three-sided pocket on the WD40 surface (Fig. 10a). This is
clearly important here as is shown by the epitope mapping
data (STD-NMR) where Leu45 and Ile46 hydrophobic side
chains along with negative charged (Asp, pSer and Glu) side
chains contact the site. The fact that a Leu or Ile was close to
the known DpSGXXpS binding fragment enhanced interaction
of the Vpu ligand to b-TrCP protein.
4.2.2. The DpSG motif in the binding site of the b-TrCP as WD
domain protein
The C-terminal domain of b-TrCP contains seven WD repeats
(Fig. 2b), known to form interfaces for protein–protein
interaction [27], and required for optimal binding to Vpu.
Modeling of the F-box protein b-TrCP [52] reveals an extensive
basic region on the front face of the propeller, which may
engage substrate phosphoepitopes. The b-TrCP WD domain
has the ability to recognize pSer epitopes in the context of the
peptides 27 (2006) 194–210
207
Fig. 10 – A portion of the seven b-propeller structure of the WD40 repeat region of the yeast F-box protein b-TrCP is
shown based on the crystal structure of the complex (b-TrCP-SkP1—b-catenin peptide) [50]. The residues of the protein
surface are colored in yellow according to positive electrostatic potential and hydrogen bonding. (a) Superimposition
of the DpSG fragment from P1 peptide (in orange) and the X-ray crystal structure of the b-Catenin peptide (in green) also
highlights similarities for the b-TrCP combining site protein between Vpu and b-catenin protein concerning the first
phosphate group of pSer residue of the DpSGXXpS bound motif, pSer52 and pSer33. Interestingly, Leu45 and Ile46, whose
hydrophobic nature is conserved in the P1 peptide fragment, made van der Waals contacts with a hydrophobic pocket
composed of the aliphatic portion of the Phe523, and Val516 (WD7) side chains in b-TrCP (in blue). (b) Superimposition of
the DpSG fragment from P2 peptide (in magenta) and the X-ray crystal structure of the b-catenin peptide (in green)
highlights similarities for the b-TrCP combining site protein between Vpu and b-catenin protein concerning the first
phosphate group of pSer residue of the DpSGXXpS bound motif. pSer52 and pSer33 are able to make electrostatic interaction
with the positive Arg285 (WD1) and some hydrogen bonds with the side chain hydroxyl groups of Tyr271, Ser309 and
Ser325 (WD1), while pSer56 and pSer37 have a different location, pSer56 is close to Lys365 (WD3) and pSer37 to Arg431
(WD5). A second broader site is formed by a positive charge distribution on the surface (Arg367, Arg390, Arg431 and Lys365)
able to bind the second pSer. (For interpretation of the references to color in this figure legend, the reader is referred to the
web version of the article.)
208
peptides 27 (2006) 194–210
adjacent Gly residue while other WD domain employs a pThr–
Pro peptide [26,29]. The Gly or the Pro binding pocket is able to
accommodate other residues with a same propensity to form b
turns. The specificity of phosphorylation-recognition by the
WD domain of b-TrCP is characterized by a dedicated pSer–Gly
binding pocket that selects residues N-terminal to the
phosphorylation site.
The X-ray crystallographic analysis of b-TrCP protein
complexed with a fragment b-Catenin substrate peptide [50]
reveals the binding site specific for a phosphopeptide complex
bound to an seven-bladed WD40 propeller domain (Fig. 9a),
and that nearly all of the b-TrCP contacts are made by the six
residue pSGIHpS motif. However, the fragment in the complex
is too short to highlight a hydrophobic interaction upstream
(Fig. 9b).
Interaction of HIV-1 Vpu with b-TrCP relies on motif
DpSGNEpS similar to that found in the other substrates of bTrCP (IkBa and b-Catenin). It is interesting to note that the first
serine residue of this motif seems to play an essential role in
this interaction. The second serine residue is involved in the
case of Vpu and b-Catenin but not required for the interaction
of ATF4 with b-TrCP (ATF4, a member of the family of
transcription factors). Interaction of ATF4 with b-TrCP relies
on motif DSGXXXS [18].
The P2 bound structure corresponds to the model’s
prediction of substrate recognition by the b-TrCP1 WD40
domain. b-TrCP could associate with P2 peptide via chargebased interactions. In the P2 peptide, except for Gly53, all the
chain residues, in the EDpSGNEpSE bend form a negatively
charged surface that would provide a plausible binding region
in contact with the positive protein b-TrCP surface. The WD
domain of b-TrCP has positive amino acids, Arg and Lys
(Fig. 10b) that could accept the diphosphorylated segment,
DpSGXXpS present in the Vpu, b-Catenin and IkBa protein.
In Vpu, the phosphate group of pSer52 is able to make the
largest number of contacts, in forming direct hydrogen bond
with the side chain hydroxyl groups of Tyr271, Ser309 and
Ser325 in the WD domain of b-TrCP protein. The phosphate
group forms also direct electrostatic interactions with the
guanidium group of Arg285 (Fig. 10b). This creates a hydrogen
bond network in the protein around the phosphate group, and
can explain the high saturation transfer towards the phosphate group of pSer52. On the basis of previous mutational
analysis [26,36,41], the Arg residues are essential for function.
Study of mutations made it possible to identify the binding site
of b-Catenin and IkBa for b-TrCP [54]. The three Arg285Glu,
Arg474Glu and Arg521Glu mutations block the interaction
with b-Catenin.
Asp51, which is an invariant binding motif residue, is also
able to make an extensive contact as its side chain allows a
hydrogen bond with Arg521, Arg474 and Tyr271 in the WD
domain of b-TrCP protein. The Asp51 residue gives the higher
relative saturation transfer (STD signal 100%) for the two P1
and P2 bound peptides (Fig. 5) corresponding to a tight contact
to the b-TrCP protein surface.
Gly53, also an invariant binding motif residue, is able to
pack with the b-TrCP receptor in an environment with little
space for a non-glycine residue. The conservation of the Gly53
residue is also justified by the need for compact side chains not
to disturb the position of Leu331 and Leu351 of b-TrCP protein.
The position of the XX residues in the DpSGXXpS motif,
located above the central channel of the seven-bladed WD40
propeller domain and, without particular implication of the
side chains, can explain the variability of the residues to these
positions in the phosphorylation motif.
The second pSer, is less embedded what can explain its role
perhaps less essential in the interaction, but its original
orientation in the bound Vpu can explain a better interaction
of this ligand (Fig. 10b). The binding site seems to be enough
restricted to select the DpSG motif (Arg285, Arg474, Arg521 and
some hydrogen bonds) since this part of the motif is relatively
hidden and a second broader site formed by a positive charge
distribution on the surface (Arg367, Arg390, Arg431 and
Lys365) laid out around the second pSer. The multiplicity
and the proximity of these charges can also explain the
existence of longer phosphorylation motifs (DpSGXXXpS or
DpSGXXXXpS). The lengthening of the phosphorylation motif
introduced a great mobility of the terminal part of the motif,
what can attenuate the affinity of the ligand for the receptor.
Nevertheless, it is possible that the second pSer carries out an
electrostatic interaction with one of the other positive charges
accessible on the b-TrCP protein surface. This positive charge
distribution can also highlight on the best affinity of P-Vpu
compared to the other ligands. Indeed P-Vpu is the only
protein containing a negative charge (Glu) after the second
pSer among the other ligands (Fig. 10b). Its implication in the
binding is consistent with the STD-NMR data (Fig. 5) and the
variation of the chemical shift of the Glu57 residue (Fig. S11 in
Supplementary data). The negative potential generated by the
phosphorylated Ser52 and Ser56 increases with Glu57 (and
Glu55). A second negative pole around pSer56 reinforces the
Vpu binding to the b-TrCP protein, which was mainly
characterized by the first pSer, pSer52 and Asp51 in close
proximity.
5.
Conclusion
The approaches discussed in this study allow screening of
compounds as well as a detailed identification of the
fragments involved in the binding events. The Vpu analog
peptides, P1 and P2 were characterized efficiently by NMR to
bind to the b-TrCP WD domain. The two peptides exhibit
different secondary structure characteristics in interaction
with b-TrCP. P2 exhibits a structure with a large bend at
DpSGNEpS and P1 exhibits a turn type of structure at IERAED.
The differences in the structures of peptides observed may
contribute to the selectivity of the b-TrCP receptor for Vpu
analog peptides. In conclusion, we have shown that interaction in b-TrCP protein of Vpu analogs leads to different
possible structures but with the DpSG fragment conformation
very conserved. A different orientation of the second pSer for
Vpu which is the only protein with the presence of one Glu
residue containing a negative charge after the second pSer
emphasizes the interaction with the b-TrCP protein, and this
can also highlighted on the best affinity of P-Vpu compared to
the other ligands. These structural differences may be
important for the development of novel b-TrCP receptor
selective (ligands or inhibitors). This study indicates the
important structural features responsible for a ligand’s ability
peptides 27 (2006) 194–210
to bind b-TrCP receptor, and define a structural DpSG motif
that binds the b-TrCP receptor, a target of much biological
interest.
Acknowledgements
This work was supported by grants from the organizations
ANRS (Agence Nationale pour la recherche contre le SIDA),
SIDACTION, ARC and Ligue Nationale contre le Cancer.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at 10.1016/j.peptides.2005.07.018.
Appendix B. Supplementary data
Table S4 listing 1H and 13C NMR Chemical Shifts of the Free
Peptides P1 and P2 and Table S5 listing some inter-residue
1
H–1H distances calculated from unambiguous NOE volumes
for P1 and P2 bound peptides; Fig. S11 showing difference for
the residues of P1 and P2 between the chemical shift of a given
resonance free in buffer solution and the chemical shift in the
presence of the GST–b-TrCP protein, for NH, Ha, Ca and Cb
resonances. This material is available free of charge via the
Internet at http://www.sciencedirect.com.
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